CN110885846B - Microorganism for synthesizing baicalein and scutellarin, preparation method and application thereof - Google Patents

Abstract

The invention relates to a microorganism for synthesizing baicalein and scutellarein, a preparation method and application thereof. The present inventors modified the heterologous metabolic pathway of the host cell by means of genetic engineering, and obtained an engineering strain with high production of baicalein and scutellarein. The invention also provides a process for producing scutellarein and scutellarein by using the engineering strain.

Description

Microorganism for synthesizing baicalein and scutellarein, its preparation method and application

technical field

The invention relates to the technical fields of synthetic biology and medicine, in particular, the invention relates to microorganisms for synthesizing scutellarein and scutellarein, a preparation method and application thereof.

Background technique

Scutellaria Baicalensis Georgi is a well-known traditional medicine in China. It is a plant of the family Labiatae. The traditional Chinese medicine Scutellaria Baicalensis is the dried root of the plant Scutellaria baicalensis. It has a long history of medicinal use and can be used for the treatment of wind-heat, damp-heat and other diseases. Erigeron breviscapus is the dried whole herb of the Compositae plant Erigeron breviscapus. It is cold in nature and bitter in taste. Scutellaria baicalensis and Erigeron breviscapus extracts have long been widely used in traditional Chinese medicine preparations. The main raw materials of Yinhuang Tablets, Shuanghuanglian Oral Liquid and Lancen Oral Liquid are all Scutellaria baicalensis extracts. The main active ingredient of traditional Chinese medicine Qingkailing is baicalein. It has the functions of anti-inflammation, prevention and treatment of diarrhea, liver disease and tumors; common breviscapine formulations include breviscapine tablets and breviscapine oral liquid, etc., and their main form of metabolism and absorption in the organism is its aglycone scutellarein. Therefore, both baicalein and scutellarein have certain new drug development value.

Baicalein and scutellarein are two structurally similar and important flavonoids. The molecular formula of baicalein is C ₁₅ H ₁₀ O ₅ and the molecular weight is 270.24, while the molecular weight of scutellarein is C ₁₅ H ₁₀ O ₆ and the molecular weight is 286.24. Their structures are shown in Figure 1.

Like most natural products of plant origin, baicalein and scutellarein are mainly prepared by chemical synthesis and organic solvent extraction. Organic solvent extraction is mainly to extract the tissues of medicinal plants such as Scutellaria baicalensis, Erigeron breviscapus, and Scutellaria barbata. In this process, a large amount of organic solvents are required, and there are also problems such as cumbersome separation processes and high industrialization costs. The most important thing is that this method is subject to difficult problems such as slow plant growth and damage to medicinal resources. Although baicalein and scutellarein can also be obtained in large quantities through chemical synthesis, the raw materials involve chemical substances such as cinnamic acid or its derivatives, oxyphenol, etc., which limits its application in the fields of medicine and food to a certain extent. Moreover, problems such as the use of toxic reagents and expensive chemical catalysts are also involved in the synthesis process.

Synthetic biology is based on rational design, integrating and assembling standardized biological components to construct artificial life systems with excellent performance. Once synthetic biology was born, its ideas and designs have profoundly influenced the development of industrial microbial technology, enabling microbial technology to play a greater role in the development and production of drugs, biofuels, and fine chemicals.

In the art, synthetic elements of various natural products have been assembled to enable heterologous synthesis in microorganisms. However, the two active flavonoids, baicalein and scutellarein involved in the present invention, have not yet been reported to be successfully heterologously synthesized in microorganisms. Therefore, it is urgent to construct a microbial strain capable of heterologously synthesizing baicalein and scutellarein.

Contents of the invention

The object of the present invention is to provide microorganisms for synthesizing baicalein and scutellarein, their preparation method and application.

In a first aspect of the present invention, a method for producing baicalein and scutellarein is provided, comprising: (1) introducing and expressing flavone 6-hydroxylase (F6H) and cytochrome P450 oxidoreductase (CPR) in host cells ) gene, and chrysin or apigenin synthesis gene; and (2) cultivating the host cell in a culture system containing phenylalanine and/or tyrosine, thereby producing baicalein or scutellarein.

In a preferred example, the chrysin or apigenin synthesis gene includes: expression of phenylalanine ammonia-lyase (phenylalanine ammonia-lyase, PAL), 4-coumarate coenzyme A ligase (4-coumarate: CoA ligase, 4CL), chalcone synthase (chalcone synthase, CHS), chalcone isomerase (chalcone isomerase, CHI) and flavone synthase I (flavone synthase I, FNSI) genes; When introduced into host cells, the genes expressing phenylalanine ammonia lyase, 4-coumaric acid coenzyme A ligase, chalcone synthase, chalcone isomerase and flavone synthase I exist in the same expression in the carrier.

In another preferred example, the flavone 6-hydroxylase is derived from Scutellaria baicalensis, including its homologues (homologous genes or polypeptides from other species); the CPR is derived from Arabidopsis Mustard (Arabidopsis thaliana), also including its homologues.

In another preferred example, the PAL is derived from Rhodotorula toruloides (Rhodotorula toruloides), also including its homologues; the 4CL is derived from parsley (Petroselium crispum), also including its homologues; the The CHS derived from petunia (Petunia X hybrida) also includes its homologue; the CHI derived from alfalfa (Medicagosativa) also includes its homologue; the FNS I derived from parsley (Petroselium crispum ), including its homologues.

In another aspect of the present invention, there is provided a method for producing baicalein and scutellarein, comprising: (1) introducing and expressing flavone 6-hydroxylase (F6H) and cytochrome P450 oxidoreductase (CPR) in host cells ) to obtain a recombinant host cell; and (2) cultivating the recombinant host cell in a culture system containing chrysin or apigenin, thereby producing baicalein or scutellarein.

In another aspect of the present invention, there is provided a method for converting chrysin or apigenin into baicalein or scutellarein: catalyzing chrysin or apigenin with flavone 6-hydroxylase and cytochrome P450 oxidoreductase, thereby Add a hydroxyl group to the structure of chrysin or apigenin to form baicalein or scutellarein.

In a preferred example, the flavone 6-hydroxylase (F6H) is a mutant flavone 6-hydroxylase that truncates amino acids at positions (1-10) to (20-30) of the N-terminus; preferably , is a mutant flavone 6-hydroxylase that truncated amino acids at positions (2-5)-(22-28) of the N-terminus.

In another preferred example, the flavone 6-hydroxylase is fused with a polypeptide tag, and the polypeptide tag is selected from: calf serum 17 hydroxylase N-terminal 8 amino acid polypeptide (8RP), small molecule ubiquitin modified Related protein (Sumo), maltose binding protein (MBP), cytochrome P450 2B1 family soluble protein (2B1), or combination thereof; preferably maltose binding protein or cytochrome P450 2B1 family soluble protein, or combination thereof; preferably The polypeptide tag is located at the N-terminus.

In another preferred example, the cytochrome P450 oxidoreductase (CPR) is a mutant cytochrome P450 oxidoreductase that truncated amino acids at positions (1-20)-(60-85) of the N-terminal; Preferably, it is a mutant cytochrome P450 oxidoreductase that truncates (2-10)-(65-80) amino acids at the N-terminus; more preferably, it is a truncated N-terminus (2-5)-(70 -75) amino acid mutant cytochrome P450 oxidoreductase.

In another preferred example, the host cells include: prokaryotic cells or eukaryotic cells; preferably, the prokaryotic cells include: Escherichia coli cells, Bacillus subtilis cells; the eukaryotic cells include: yeast cell.

In another aspect of the present invention, a recombinant host cell is provided, which includes exogenous genes expressing flavone 6-hydroxylase and cytochrome P450 oxidoreductase.

In another preferred example, the recombinant host cell further includes an exogenous chrysin or apigenin synthesis gene.

In another preferred example, the polypeptide tag is a single copy or a sequence structure of 2-10 copies (such as 3, 4, 5, 6, 8 copies) in series.

In another aspect of the present invention, the use of any one of the aforementioned recombinant host cells is provided for producing scutellarein and scutellarein.

In a preferred example, for the bacterial strain that does not have chrysin or apigenin synthesis gene in the cell, it is used to produce baicalein and scutellarein with chrysin or apigenin added by exogenous sources; for the presence of chrysin or apigenin in the cell A strain with an apigenin synthesis gene for producing baicalein and scutellarein under the culture conditions of exogenously added phenylalanine and/or tyrosine.

In another aspect of the present invention, there is provided a method for preparing host cells for producing baicalein and scutellarein, comprising: introducing genes expressing flavone 6-hydroxylase and cytochrome P450 oxidoreductase into the host cells , to obtain a recombinant strain; preferably, it also includes introducing a chrysin or apigenin synthesis gene.

In another aspect of the present invention, there is provided a kit for producing scutellarein and scutellarein. The kit includes any of the aforementioned recombinant host cells.

In another preferred example, the kit further includes: host cell culture medium, instructions for use, and the like.

In another aspect of the present invention, mutant flavone 6-hydroxylase is provided, which corresponds to wild-type flavone 6-hydroxylase (F6H), truncating the N-terminal (1-10)-(20-30) Amino acid; preferably, amino acids (2-5)-(22-28) of the N-terminal are truncated; preferably, it has the amino acid sequence shown in SEQ ID NO:2.

In another aspect of the present invention, a mutant cytochrome P450 oxidoreductase is provided, which corresponds to the wild-type cytochrome P450 oxidoreductase, and amino acids at positions (1-20) to (60-85) of the N-terminal are truncated; Preferably, amino acids at positions (2-10) to (65-80) of the N-terminus are truncated; more preferably, amino acids at positions (2-5) to (70-75) of the N-terminus are truncated; preferably, the It has the amino acid sequence shown in SEQ ID NO:8.

In another aspect of the present invention, a fusion polypeptide is provided, which includes any of the aforementioned mutant flavone 6-hydroxylases, and a polypeptide tag fused thereto, and the polypeptide tag is selected from: 8RP, Sumo, MBP , 2B1; preferably MBP or 2B1.

In a preferred example, the fusion polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

In another aspect of the present invention, a polynucleotide is provided, which encodes: the mutant flavone 6-hydroxylase; or the mutant cytochrome P450 oxidoreductase; or the fusion polypeptide.

In another aspect of the present invention, an expression construct is provided, which includes: any polynucleotide described above; or encodes any mutant flavone 6-hydroxylase described above or the above-mentioned The polynucleotide of the fusion protein, and the polynucleotide encoding the aforementioned mutant cytochrome P450 oxidoreductase.

In another preferred example, the expression construct further includes a promoter and a terminator operably linked to the above-mentioned polynucleotide.

In another aspect of the present invention, there is provided the use of the mutant flavone 6-hydroxylase or the fusion protein, and the mutant cytochrome P450 oxidoreductase, which are used in the structure of chrysin or apigenin Add a hydroxyl group to form baicalein or scutellarein.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. Structural formulas of baicalein and scutellarein.

Figure 2. Biosynthetic pathways of baicalein and scutellarein.

Fig. 3. Schematic diagram of the construction of plasmid pYH66.

Fig. 4. Schematic diagram of the construction of plasmid pYH57.

Fig. 5. HPLC detection patterns of engineering strain BL21(DE3)-pYH57-pYH66 and standard product baicalein. Among them, i represents BL21(DE3)-pETDuet-1-pCDFDuet-1 fermentation broth as a blank control; ii represents BL21(DE3)-pYH57-pYH66 fermentation broth added with phenylalanine; iii represents baicalein standard.

Fig. 6. The mass spectrogram of baicalein produced by engineering strain BL21(DE3)-pYH57-pYH66.

Fig. 7. HPLC detection pattern of engineering strain BL21(DE3)-pYH57-pYH66 and standard product scutellarein. Among them, i represents BL21(DE3)-pETDuet-1-pCDFDuet-1 fermentation liquid as a blank control; ii represents tyrosine-added fermentation liquid of BL21(DE3)-pYH57-pYH66; iii represents scutellarein standard.

Fig. 8. The mass spectrum of scutellarein produced by engineering strain BL21(DE3)-pYH57-pYH66.

Figure 9. SbF6H and AtCPR mutants catalyze the production of baicalein from chrysin.

A, schematic diagram of the key elements in the constructed plasmid;

B, in recombinant Escherichia coli, the conversion rate of catalyzing chrysin to generate baicalein;

C, HPLC analysis of recombinant Escherichia coli catalytic reaction solution. Among them, Chr means chrysin, and Bai means baicalein.

Detailed ways

The present inventor is committed to using heterologous synthesis of baicalein and scutellarein in microorganisms, and improving the yield of biological production of baicalein and scutellarein. Transformation, obtained high-yielding baicalein and wild scutellarein engineering strains.

As used herein, the "N-terminal (1-10)-(20-30) amino acid" refers to any amino acid starting from the 1-10th position from the N-terminal and ending from the N-terminal Any amino acid in the 20th-30th position.

As used herein, the "N-terminal (2-5)-(22-28)" refers to any amino acid starting from the 2-5th position from the N-terminal and ending at the 2-5th amino acid from the N-terminal Any amino acid in positions 22-28.

As used herein, the "N-terminal (1-20) ~ (60-85) position" refers to any amino acid starting from the 1-20th position from the N-terminal and ending at the 1st-20th amino acid from the N-terminal Any amino acid in positions 60-85.

As used herein, the "N-terminal (2-10) ~ (65-80) position" refers to any amino acid starting from the 2-10th position from the N-terminal and ending at the 2-10th amino acid from the N-terminal Any amino acid in positions 65-80.

As used herein, the "N-terminal (2-5) ~ (70-75) position" refers to any amino acid starting from the 2-5th position from the N-terminal and ending at the 2-5th amino acid from the N-terminal Any amino acid in positions 70-75.

As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources.

As used herein, the term "operably linked (linked)" or "operably linked (linked)" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed at a specific position relative to the nucleic acid sequence of the target gene, so that the transcription of the nucleic acid sequence is guided by the promoter region, thus, the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, the "expression construct" refers to a recombinant DNA molecule comprising the desired nucleic acid coding sequence, which may comprise one or more gene expression cassettes. Said "construct" is usually contained in an expression vector.

As used herein, the PAL, 4CL, CHS, CHI and FNSI proteins are proteins that form the synthesis pathway of chrysin or apigenin in the expression system.

As used herein, the F6H and CPR proteins are proteins that convert chrysin or apigenin into baicalein or scutellarein in an expression system.

The above-mentioned wild-type proteins or genes have been identified in the art, and therefore, can be obtained and prepared from public sources. As a preferred mode of the present invention, PAL is derived from Rhodiola rosea (Rhodotorula toruloides), which has the sequence shown in GenBank accession number AAA33883.1; 4CL is derived from parsley (Petroselium crispum), which has the sequence shown in GenBank accession number KF765780.1. CHS is derived from petunia (Petunia X hybrida), which has the sequence shown in GenBank accession number KF765781.1; CHI gene is derived from alfalfa (Medicago sativa), which has the sequence shown in GenBank accession number KF765782.1 Sequence; FNS I is derived from parsley (Petroselium crispum), which has the sequence shown in Swiss-Prot accession number Q7XZQ8.1.

Wild-type F6H and CPR have also been identified in the art. As a preferred embodiment of the present invention, F6H is derived from Scutellaria baicalensis, which has the sequence shown by GenBank accession number ASW21050.1. As a preferred mode of the present invention, CPR comes from Arabidopsis thaliana, which has the sequence shown in GenBank accession number NP_849472.2.

The inventors found that in the process of using host cells to produce baicalein and scutellarein, only a small amount of products can be produced by using wild-type F6H, and large-scale production cannot be realized. Therefore, multiple proteins involved in the reaction were modified. After a large number of screening and analysis, some optimal transformation schemes were obtained, which greatly improved the production of microorganisms, especially prokaryotic expression systems such as scutellarein and scutellarein in Escherichia coli.

Therefore, as a preferred mode of the present invention, a mutant F6H is provided, which corresponds to the wild-type F6H, and amino acids at positions (1-10) to (20-30) of the N-terminal are truncated; preferably, the N-terminal truncated (2-5)-(22-28) amino acids at the terminal; more preferably, amino acids 2-25 at the N-terminus are truncated.

As a preferred mode of the present invention, there is also provided a fusion protein containing the F6H or mutant F6H, which includes F6H or any mutant F6H, and a polypeptide tag fused thereto, and the polypeptide tag is selected from: 8RP, Sumo, MBP, 2B1, or a combination thereof; preferably MBP or 2B1. The polypeptide tag and the F6H or mutant F6H may or may not contain a connecting peptide, and the connecting peptide does not affect the biological activity of the two.

As a preferred mode of the present invention, a mutant CPR is provided, which corresponds to the wild-type CPR, and amino acids (1-20) to (60-85) of the N-terminus are truncated; (2-10)-(65-80) amino acids; more preferably, the N-terminal (2-5)-(70-75) amino acids are truncated.

On the basis of the above-mentioned preferred proteins (including the above-mentioned wild-type proteins and mutant proteins), the present invention also includes their fragments, derivatives and analogs that retain biological activity. Their protein fragments, derivatives or analogs can be several (usually 1-50, more preferably 1-20, even more preferably 1-10, 1-5, 1-3, or 1-2) deletion, insertion and/or substitution of amino acids, and addition or deletion of one or several (for example, within 100, within 80, within 50, within 20 at the C-terminal and/or N-terminal, relatively Preferably within 10, more preferably within 5) amino acids. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. However, for the above-mentioned mutant proteins, in their further variant forms, the above-mentioned N-terminal truncation is carried out.

On the basis of the above-mentioned preferred proteins (including the above-mentioned wild-type proteins and mutant proteins), the present invention also includes their analogs. The difference between these analogs and the natural protein may be the difference in amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.

On the basis of the above-mentioned preferred proteins (including the above-mentioned wild-type proteins and mutant proteins), the present invention also includes proteins with high homology (such as 70% homology with the specific protein sequence listed) % or higher; preferably homology is 80% or higher; more preferably homology is 90% or higher, such as homology 95%, 98% or 99%), and has the same function as the corresponding polypeptide The proteins are also included in the present invention.

Proteins or genes from specific species are listed in the present invention. It should be understood that although proteins or genes obtained from specific species are preferably studied in the present invention, proteins or genes obtained from other species are highly homologous (such as having more than 60%, such as 70%, 80%, 85% , 90%, 95%, or even 98% sequence identity) other proteins or genes are also within the scope of the present invention.

The invention also relates to the invention also provides a polynucleotide sequence encoding the protein of the invention or its conservative variant protein. A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The polynucleotide encoding the mutant mature protein of the present invention includes: the coding sequence encoding only the mature protein; the coding sequence of the mature protein and various additional coding sequences; the coding sequence of the mature protein (and optional additional coding sequences) and non- coding sequence.

The present invention also includes the polynucleotide sequence formed by codon-optimizing the sequence of the gene, for example, codon-optimizing according to the preference of the host cell.

In the present invention, a high-yielding baicalein and scutellarein engineering strain is also constructed, which includes exogenously introduced expression F6H (especially the mutant F6H or fusion protein) and CPR (especially the mutant F6H or fusion protein) genes. The recombinant bacterial strain is cultivated, and chrysin or apigenin is added into the culture system, so as to produce baicalein or scutellarein.

In the present invention, another kind of high-yielding baicalein or scutellarein engineering strain is also constructed, which includes the expression of F6H (especially the mutant F6H or fusion protein) and CPR (especially the mutant F6H or fusion protein) gene, and chrysin or apigenin synthesis gene. The chrysin or apigenin synthesis genes include: genes expressing PAL, 4CL, CHS, CHI and FNSI proteins.

The bacterial strain of the invention has good stability, and can realize large-scale cultivation and production of baicalein or scutellarein in a bioreactor. The yield of baicalein or scutellarein in the preferred strain of the present invention is very high.

In the present invention, Escherichia coli produces baicalein or scutellarein to realize more economical and convenient manufacture of baicalein or scutellarein.

The invention also provides a kit for producing baicalein or scutellarein engineering strains. In addition, it may also include Escherichia coli culture medium, baicalein or scutellarein separation or detection reagents, instructions for use, and the like.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to the conditions described in J. Sambrook et al., edited by J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the conditions described in the manufacturer suggested conditions.

Experimental Materials

AxyPrep Total RNA Miniprep Kit, Polymerase Chain Reaction (PCR) Gel Recovery Kit, and Plasmid Extraction Kit are all products of Axygen in the United States; PrimeScript RT reagent Kit with gDNA Eraser (PerfectReal Time) Polymerase Kit, Polymerase The chain reaction (PCR) high-fidelity enzyme PrimeSTAR Max DNAPolymerase is a product of TAKARA; the restriction endonucleases are all NEB products.

Escherichia coli DH10B was used for gene cloning, and Escherichia coli BL21 (DE3) strain was used for protein expression and production of baicalein and wild scutellarein. The pET28a, pEDDuet-1, pCDFDuet-1 vectors were used for metabolic pathway gene assembly.

The standard compounds, baicalein and scutellarein, were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Other reagents were domestic analytically pure or chromatographically pure reagents, which were purchased from Sinopharm Chemical Reagent Co., Ltd.

Arktik Thermal Cycler (Thermo Fisher Scientific) was used for PCR; ZXGP-A2050 constant temperature incubator and ZWY-211G constant temperature culture shaker were used for constant temperature culture; 5418R high-speed refrigerated centrifuge and 5418 small centrifuge (Eppendorf) were used for centrifugation. Concentrator plus concentrator (Eppendorf) was used for vacuum concentration; OD ₆₀₀ was detected by UV-1200 ultraviolet-visible spectrophotometer (Shanghai Meipuda Instrument Co., Ltd.). The rotary evaporation system consists of IKARV 10digital rotary evaporator (IKA), MZ 2C NT chemical diaphragm pump, and CVC3000 vacuum controller (vacuubrand). For high performance liquid chromatography, a Dionex UltiMate 3000 liquid chromatography system (Thermo Fisher Scientific) was used.

Liquid phase detection conditions: A phase: 0.1% formic acid water, B phase: acetonitrile; separation conditions: 0-20min 20% B phase-55% B phase, 20-22min 55% B phase-100% B phase, 22-27min 100% Phase B, 27-35min100% Phase B-20% Phase B, 35-40min, 20% Phase B; detection wavelength: 340nm, column temperature: 30°C. Chromatographic column: Thermo syncronis C18 reverse phase column (250mm×4.6mm, 5μm).

Embodiment 1, polypeptide and its sequence optimization

1. Optimization and transformation of F6H polypeptide sequence

F6H (SbF6H) from Scutellaria baicalensis is 517aa in length (Genbankaccess no.ASW21050.1), and the specific sequence is as follows (SEQ ID NO: 1):

M ELSSVIYGAIALLSLFYCYLHFSK PKKSSLNAPPEAGGARFITGHLHLMDGRSASDKLPHINLGLLADQHGPIFTIRLGVHRAVVVSSWELAKEIFTTHDTAVMARPRLIADDYLSYDGASLGFSPYGPYWREIRKLVTTELLSARRIELQRATRVREITQFTGELYKLWEEKKDGSGRVLVDMKQWLGN LSLNLVSRMVVGKRFYGGDDSETTKRWRGVMREFFQLIGQFIPGDGLPFLRWLDLGGFEKRTRDTAYELDKIIAMWLAEYRKREYSGDDKEQCFMALMLSLVQANPTLQLHYDADTIIKATCQVLISAASDTTTVILIWVISLLLNNADVLKKVQEELDEQVGRERRVEESDISNLPY LQAVVKETMRLYPPAPFAGVRAFSEDCTVGGYHIQKGTFLIVNLWKLHRDPRVWSDDALEFKPQRFFDKKVEVKGQDFELMPFGGGRRMCPGSSNLGMHMVHFVLANILQAFDITTGSTVDMTESVGLTNMKATPLDAILTPRLSPTLY*

Modification 1: The inventors modified the sequence of SEQ ID NO: 1, removed amino acids 2-25, and added two amino acids of MA to the N-terminal to obtain an improved F6H mutant trF6H. The specific sequence is as follows (SEQ ID NO :2):

MA MPKKSSLNAPPEAGGARFITGHLHLMDGRSASDKLPHINLGLLADQHGPIFTIRLGVHRAVVVSSWELAKEIFTTHDTAVMARPRLIADDYLSYDGASLGFSPYGPYWREIRKLVTTELLSARRIELQRATRVREITQFTGELYKLWEEKKDGSGRVLVDMKQWLGNLSLNLVSRMVVGKRFYGGDDSET TKRWRGVMREFFQLIGQFIPGDGLPFLRWLDLGGFEKRTRDTAYELDKIIAMWLAEYRKREYSGDDKEQCFMALMLSVQANPTLQLHYDADTIIKATCQVLISAASDTTTVILIWVISLLLNNADVLKKVQEELDEQVGRERRVEESDISNLPYLQAVVKETMRLYPPAPFAGVRAF SEDCTVGGYHIQKGTFLIVNLWKLHRDPRVWSDDALEFKPQRFFDKKVEVKGQDFELMPFGGGRRMCPGSNLGMHMVHFVLANILQAFDITTGSTVDMTESVGLTNMKATPLDAILTPRLSPTLY*

Modification 2: The inventors modified the sequence of SEQ ID NO: 1, removed amino acids 2-25, and added the amino acid sequence of 8RP to the N-terminal to obtain an improved F6H mutant 8RPtrF6H, the specific sequence of which is as follows (SEQ ID NO:3):

MALLLAVF MPKKSSLNAPPEAGGARFITGHLHLMDGRSASDKLPHINLGLLADQHGPIFTIRLGVHRAVVVSSWELAKEIFTTHDTAVMARPRLIADDYLSYDGASLGFSPYGPYWREIRKLVTTELLSARRIELQRATRVREITQFTGELYKLWEEKKDGSGRVLVDMKQWLGNLSLNLVSRMVVGKRFYG GDDSETTKRWRGVMREFFQLIGQFIPGDGLPFLRWLDLGGFEKRTRDTAYELDKIIAMWLAEYRKREYSGDDKEQCFMALMLSVQANPTLQLHYDADTIIKATCQVLISAASDTTTVILIWVISLLLNNADVLKKVQEELDEQVGRERRVEESDISNLPYLQAVVKETMRLYPPAPFA GVRAFSEDCTVGGYHIQKGTFLIVNLWKLHRDPRVWSDDALEFKPQRFFDKKVEVKGQDFELMPFGGGRRMCPGSNLGMHMVHFVLANILQAFDITTGSTVDMTESVGLTNMKATPLDAILTPRLSPTLY*

Modification 3: The inventors modified the sequence of SEQ ID NO: 1, removed amino acids 2-25, and added the amino acid sequence of Sumo to the N-terminal to obtain an improved F6H mutant SumotrF6H, the specific sequence of which is as follows (SEQ ID NO:4):

MADSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYD GIRIQADQTPEDLDMEDNDIIEAHREQIGGMPKKSSLNAPPEAGGARFITGHLHLMDGRSASDKLPHINLGLLADQHGPIFTIRLGVHRAVVVSSWELAKEIFTTHDTAVMARPRLIADDYLSYDGASLGFSPYGPYWREIRKLVTTELLSARRIELQRATRVREITQFTGELYKLWEEKKDGSGRVLVDMKQWLGNLSLNLVSRMVVGKRFYGGDDSETTK RWRGVMREFFQLIGQFIPGDGLPFLRWLDLGGFEKRTRDTAYELDKIIAMWLAEYRKREYSGDDKEQCFMALMLSVQANPTLQLHYDADTIIKATCQVLISAASDTTTVILIWVISLLLNNADVLKKVQEELDEQVGRERRVEESDISNLPYLQAVVKETMRLYPPAPFAGVRAFSEDC TVGGYHIQKGTFLIVNLWKLHRDPRVWSDDALEFKPQRFFDKKVEVKGQDFELMPFGGGRRMCPGSSNLGMHMVHFVLANILQAFDITTGSTVDMTESVGLTNMKATPLDAILTPRLSPTLY*

Modification 4: The inventors modified the sequence of SEQ ID NO: 1, removed amino acids 2-25, and added the amino acid sequence of MBP to the N-terminus to obtain an improved F6H mutant MBPtrF6H. The specific sequence is as follows (SEQ ID NO:5):

MAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDR FGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAK GKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFN KGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVN KDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTMPKKSSLNAPPEAGGARFITGHLHLMDGRSASDKLPHINLGLLADQHGPIFTIRLGVHRAVVVSSWELAKEIFTTHDTAVMARPRLIADDYLSYDGASLGFSPYGPYWREIRKLVTTELLSARRIELQRATRVREITQFTGELYKLWEEKKDGSGRVLVDMKQWLGNLSLNLVSRMVVGKRFYGGDDSETTK RWRGVMREFFQLIGQFIPGDGLPFLRWLDLGGFEKRTRDTAYELDKIIAMWLAEYRKREYSGDDKEQCFMALMLSVQANPTLQLHYDADTIIKATCQVLISAASDTTTVILIWVISLLLNNADVLKKVQEELDEQVGRERRVEESDISNLPYLQAVVKETMRLYPPAPFAGVRAFSEDC TVGGYHIQKGTFLIVNLWKLHRDPRVWSDDALEFKPQRFFDKKVEVKGQDFELMPFGGGRRMCPGSSNLGMHMVHFVLANILQAFDITTGSTVDMTESVGLTNMKATPLDAILTPRLSPTLY*

Modification 5: The inventors modified the sequence of SEQ ID NO: 1, removed amino acids 2-25, and added the amino acid sequence of 2B1 to the N-terminus to obtain an improved F6H mutant 2B1trF6H. The specific sequence is as follows (SEQ ID NO:6):

MAKKTSSKGKLPPGPS MPKKSSLNAPPEAGGARFITGHLHLMDGRSASDKLPHINLGLLADQHGPIFTIRLGVHRAVVVSSWELAKEIFTTHDTAVMARPRLIADDYLSYDGASLGFSPYGPYWREIRKLVTTELLSARRIELQRATRVREITQFTGELYKLWEEKKDGSGRVLVDMKQWLGNLSLNLVSRMVV GKRFYGGDDSETTKRWRGVMREFFQLIGQFIPGDGLPFLRWLDLGGFEKRTRDTAYELDKIIAMWLAEYRKREYSGDDKEQCFMALMLSVQANPTLQLHYDADTIIKATCQVLISAASDTTTVILIWVISLLLNNADVLKKVQEELDEQVGRERRVEESDISNLPYLQAVVKETMR LYPPAPFAGVRAFSEDCTVGGYHIQKGTFLIVNLWKLHRDPRVWSDDALEFKPQRFFDKKVEVKGQDFELMPFGGGRRMCPGSSNLGMHMVHFVLANILQAFDITTGSTVDMTESVGLTNMKATPLDAILTPRLSPTLY*

2. Optimization and transformation of CPR

The length of the CPR (AtCPR) sequence from Arabidopsis thaliana (Arabidopsis thaliana) is 712aa (Genebankaccess no.NP_849472.2), specifically as follows (SEQ ID NO: 7):

MSSSSSSSTSMIDLMAAIIKGEPVIVSDPANASAYESVAAELSSMLIENRQFAMIVTTSIAVLIGCIV MLVW *

The inventors performed sequence modification on SEQ ID NO: 1, deleted amino acids 2-72, and obtained an improved AtCPR mutant trAtCPR, specifically as follows (SEQ ID NO: 8):

MRRSGSGNSKRVEPLKPLVIKPREEEIDDGRKKVTIFFGTQTGTAEGFAKALGEEAKARYEKTRFKIVDLDDYAADDDEYEEKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEGNDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDILVEQGAQRLVQVGLGDDDQCIEDD FTAWREALWPELDTILREEGDTAVATPYTAAVLEYRVSIHDSEDAKFNDINMANGNGYTVFDAQHPYKANVAVKRELHTPESDRSCIHLEFDIAGSGLTYETGDHVGVLCDNLSETVDEALRLLDMSPDTYFSLHAEKEDGTPISSSLPPPFPCNLRTALTRYACLLSSPKKSALVALAAHASDPTEAERLKHLASPAG KVDEYSKWVVESQRSLLEVMAEFPSAKPPLGVFFAGVAPRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIHKGVCSTWMKNAVPYEKSENCSSAPIFVRQSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVLFFGCRNRRMDFIYEEELQRFVESGALAELSVAFSREGPTKEY VQHKMMDKASDIWNMISQGAYLYVCGDAKGMARDVHRSLHTIAQEQGSMDSTKAEGFVKNLQTSGRYLRDVW*

Embodiment 2, the construction of the recombinant plasmid that comprises novel F6H mutant

Using pETDuet-1 as the starting plasmid, AtCPR was connected to NdeI and XhoI sites by a one-step cloning method to obtain plasmid pYH45.

Using pETDuet-1 as the starting plasmid, trAtCPR was connected to NdeI and XhoI sites by a one-step cloning method to obtain plasmid pYH46.

Further, GenScript synthesized the codon-optimized F6H coding gene sequence and ligated it into the pUC19 vector to obtain pUC19-F6H. Use F6H-F/R as the primer and pUC19-F6H as the template for PCR amplification, the system is 50 μL (PrimeSTAR Max Premix, 25 μL; the final concentration of double primers is 0.2-0.3 μM; pUC19-F6H 0.2uL; the remaining volume is sterilized with distilled water make up). PCR reaction conditions: pre-denaturation at 98°C for 2min, then denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 72°C for 20s, 25 cycles, detected by agarose electrophoresis, amplified to obtain a fragment of about 1.5kb, purified with Nco I and BamH I double enzyme digestion. The digested fragment was ligated into pYH46 digested with the same enzyme, and the ligated product was transformed into Escherichia coli DH10B competent cells. The extracted plasmid was constructed by introducing restriction sites for double digestion verification and gene sequencing to obtain the recombinant plasmid pYH59. Similarly, the digested fragment was ligated into pYH45 to obtain recombinant plasmid pYH59.

Using trF6H-F/F6H-R as primers and pUC19-trF6H as a template, the product obtained by PCR amplification was connected to the NdeI and BamH I sites of pYH46 by a one-step cloning method to obtain plasmid pYH58.

Using pUC19-trF6H as a template and 8RP-trF6H-F/F6H-R as primers to amplify, the product was connected to the NdeI and BamH I sites of pYH46 by a one-step cloning method to obtain pYH60.

Using pETSumo (purchased from Invitrogen) as a template and Sumo-F/Sumo-trF6H-R as primers to obtain a DNA fragment containing the Sumo sequence, using pUC19-trF6H as a template and Sumo-trF6H-F/F6H-R as primers to amplify The HDNA fragment containing trF6 was obtained by increasing. Using the above two DNA fragments as templates and Sumo-F/F6H-R as primers for fusion PCR, the product was ligated into the NdeI and BamH I sites of pYH46 by a one-step cloning method to obtain pYH61.

Using pMAL-c5x (purchased from Invitrogen) as a template, using MBP-F/MBP-trF6H-R as a primer to amplify the DNA fragment containing the MBP sequence, using pUC19-trF6H as a template, MBP-trF6H-F/F6H -R is a primer to amplify the DNA fragment containing trF6H, use the two DNA fragments obtained above as a template, MBP-F/F6H-R as a primer for fusion PCR, and use a one-step cloning method to connect the product to NdeI and pYH46 In the BamHI site, pYH62 was obtained.

Using pUC19-trF6H as a template and 2B1-F/F6H-R as a primer to amplify, the product was connected to the NdeI and BamH I sites of pYH46 by a one-step cloning method to obtain pYH63.

Using trF6H-F/F6H-R as primers and pUC19-trF6H as template, the product amplified by PCR was then ligated into the NdeI and BamH I sites of pYH45 by a one-step cloning method to obtain recombinant plasmid pYH64.

Using pMAL-c5x as a template and MBP-F/MBP-trF6H-R as primers to amplify the DNA fragment containing the MBP sequence, using pUC19-trF6H as a template and MBP-trF6H-F/F6H-R as primers to amplify To obtain a DNA fragment containing trF6H, use the two DNA fragments obtained above as templates and MBP-F/F6H-R as primers for fusion PCR, and then use a one-step cloning method to connect the product to the NdeI and BamH I sites of pYH45, pYH65 was obtained.

Using pUC19-2B1trF6H as template and 2B1-F/F6H-R as primer to amplify, the product was connected to NdeI and BamH I sites of pYH45 by one-step cloning method to obtain pYH66. The schematic diagram of the construction of plasmid pYH66 is shown in FIG. 3 .

The primers used in the above construction process are listed in Table 1. A schematic diagram of the key elements in the constructed plasmid is shown in Figure 9A.

Table 1

Embodiment 3, the construction of the recombinant plasmid expressing PAL, 4CL, CHS, CHI and FNSI

GenScript synthesized the PAL gene from Rhodotorula toruloides (GenBank accession number AAA33883.1), the 4CL gene from parsley (Petroselium crispum) (GenBank accession number KF765780.1), and the CHS gene (GenBank accession number KF765781.1) from morning glory (Petunia X hybrida), CHI gene (GenBank accession number KF765782.1) from alfalfa (Medicago sativa), FNS I gene from parsley (Petroselium crispum) (Swiss-Prot accession number Q7XZQ8.1), and constructed on the pET28a vector to obtain pET28-PAL, pET28-4CL, pET28-CHS, pET28a-CHI, pET28a-FNSI.

Synthesize the primers shown in Table 2. Using pET28-4CL as a template and 4CL-F-NcoI/4CL-R-BamHI as primers to obtain a PCR product, which was ligated with pCDFDuet-1 digested with NcoI/BamHI to obtain pYH40.

Using pET28-CHS as a template and CHS-F-NdeI/CHS-R-XhoI as primers to obtain a PCR product, which was ligated with NdeI/XhoI double-digested pYH40 to obtain pYH50.

Using pET28a-CHI as a template and T7CHI-F-XhoI/CHI-R-AvrII as primers to obtain a PCR product, which was connected to pYH50 to obtain pYH51.

Using pET28-PAL as a template and T7PAL-F-BamH I/PAL-R-Hind III as primers to obtain a PCR product, which was digested with BamH I/Hind III and ligated with the same double digested pYH51 to obtain plasmid pYH55.

Using pET28a-FNSI as a template and FNSI-HindIII-F/FNSI-NotI-R) as primers to obtain a PCR product, which was digested with Hind III/Not I and ligated with the same double digested pYH55 to obtain the final vector pYH57. The schematic diagram of the construction of plasmid pYH57 is shown in FIG. 4 .

Table 2

Example 4, construction and functional verification of baicalein and scutellarein synthetic strains

The schematic diagram of the biosynthesis of baicalein and scutellarein is shown in Fig. 1 .

The recombinant plasmids pYH66 and pYH57 were co-transformed into competent cells of Escherichia coli BL21(DE3) to obtain engineering strain BL21(DE3)-pYH57-pYH66.

LB solid medium (spectinomycin 80 μg/mL, ampicillin 100 μg/mL) was cultured overnight at 37°C. Pick a single clone into 2mL LB liquid medium (spectinomycin 80μg/mL, ampicillin 100μg/mL), transfer the overnight cultured bacterial liquid to a new 10mL MOPS liquid resistance medium 37°C, 250r/min culture To OD ₆₀₀ =0.5-0.6, cool the water bath to about 16°C, then add the inducer IPTG to a final concentration of 1mM, add different concentrations of sterilized phenylalanine or tyrosine and transfer to 22°C for low-temperature induction culture, in The culture was continued for 48 hours under the condition of the shaker speed of 220r/min. The BL21(DE3) recombinant strains carrying pETDuet-1 and pCDFDuet-1 empty plasmids that were not linked to foreign genes were used as blank controls, and the cultivation operation was the same as above.

At the same time, each recombinant plasmid listed in Table 2 was transformed into Escherichia coli, cultured, and the production of each product was detected.

After culturing, the conditions of expression of the compound by each recombinant strain transformed with the recombinant plasmid were detected, as shown in Table 3.

table 3

It has been verified that each recombinant strain of the present invention can successfully synthesize the target compound.

The HPLC detection patterns of the engineering strain BL21(DE3)-pYH57-pYH66 and the standard baicalein are shown in Figure 5. The mass spectrum of baicalein produced by the engineering strain BL21(DE3)-pYH57-pYH66 is shown in Figure 6.

The HPLC detection patterns of the engineering strain BL21(DE3)-pYH57-pYH66 and the standard product scutellarein are shown in Figure 7. The mass spectrum of scutellarein produced by the engineering strain BL21(DE3)-pYH57-pYH66 is shown in Figure 8.

Embodiment 5, produce with chrysin as substrate

The six recombinant plasmids pYH58-pYH66 were transformed into competent cells of Escherichia coli BL21(DE3) to obtain engineering strains, which were named BL21(DE3)-pYH58-BL21(DE3)-pYH66.

LB solid medium (ampicillin 100 μg/mL) was cultured overnight at 37°C. Pick a single clone into 2mL LB liquid medium (ampicillin 100μg/mL), transfer the overnight cultured bacterial liquid to a new 20mL MOPS liquid resistance medium at 37°C, 250r/min and cultivate until OD ₆₀₀ = 0.5-0.6 , the water bath was cooled to about 16°C, and then the inducer IPTG was added to a final concentration of 1mM, and the culture was continued for 12h under the conditions of a shaking table with a rotation speed of 220r/min and a temperature of 22°C. The above fermentation broth was centrifuged at 6000rpm, 4°C, 10min, the supernatant was removed, and the cells were collected. Resuspend to OD ₆₀₀ =30 with reaction buffer (50 mM Tris-HCl, pH 7.4, 0.1% Trixton). 1 mL of the resuspension of each recombinant bacteria was taken, 5 μL of chrysin (25 mM) and 2.5 μL of NADPH (100 mM) were added thereto, and the reaction was continued at 37° C. for 8 hours. After the reaction was completed, 10 μL of HCl (6M) and 1 mL of ethyl acetate were added to the reaction solution for extraction three times, and the residue obtained by concentrating the organic phase was dissolved in 200 μL of methanol, and 10 μL was taken for HPLC analysis.

The conversion rate of catalyzing chrysin to baicalein in each recombinant E. coli is shown in Figure 9B.

The HPLC analysis results of each recombinant Escherichia coli catalytic reaction solution are shown in Figure 9C.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

<120> Microorganisms for Synthesizing Baicalein and Scutellarein, Their Preparation Methods and Applications

<130> 186662

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<213> Scutellaria baicalensis

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Met Glu Leu Ser Ser Val Ile Tyr Gly Ala Ile Ala Leu Leu Ser Leu

1 5 10 15

Phe Tyr Cys Tyr Leu His Phe Ser Lys Pro Lys Lys Ser Ser Ser Leu Asn

20 25 30

Ala Pro Pro Glu Ala Gly Gly Ala Arg Phe Ile Thr Gly His Leu His

35 40 45

Leu Met Asp Gly Arg Ser Ala Ser Asp Lys Leu Pro His Ile Asn Leu

50 55 60

Gly Leu Leu Ala Asp Gln His Gly Pro Ile Phe Thr Ile Arg Leu Gly

65 70 75 80

Val His Arg Ala Val Val Val Ser Ser Trp Glu Leu Ala Lys Glu Ile

85 90 95

Phe Thr Thr His Asp Thr Ala Val Met Ala Arg Pro Arg Leu Ile Ala

100 105 110

Asp Asp Tyr Leu Ser Tyr Asp Gly Ala Ser Leu Gly Phe Ser Pro Tyr

115 120 125

Gly Pro Tyr Trp Arg Glu Ile Arg Lys Leu Val Thr Thr Glu Leu Leu

130 135 140

Ser Ala Arg Arg Ile Glu Leu Gln Arg Ala Thr Arg Val Arg Glu Ile

145 150 155 160

Thr Gln Phe Thr Gly Glu Leu Tyr Lys Leu Trp Glu Glu Lys Lys Asp

165 170 175

Gly Ser Gly Arg Val Leu Val Asp Met Lys Gln Trp Leu Gly Asn Leu

180 185 190

Ser Leu Asn Leu Val Ser Arg Met Val Val Gly Lys Arg Phe Tyr Gly

195 200 205

Gly Asp Asp Ser Glu Thr Thr Lys Arg Trp Arg Gly Val Met Arg Glu

210 215 220

Phe Phe Gln Leu Ile Gly Gln Phe Ile Pro Gly Asp Gly Leu Pro Phe

225 230 235 240

Leu Arg Trp Leu Asp Leu Gly Gly Phe Glu Lys Arg Thr Arg Asp Thr

245 250 255

Ala Tyr Glu Leu Asp Lys Ile Ile Ala Met Trp Leu Ala Glu Tyr Arg

260 265 270

Lys Arg Glu Tyr Ser Gly Asp Asp Lys Glu Gln Cys Phe Met Ala Leu

275 280 285

Met Leu Ser Leu Val Gln Ala Asn Pro Thr Leu Gln Leu His Tyr Asp

290 295 300

Ala Asp Thr Ile Ile Lys Ala Thr Cys Gln Val Leu Ile Ser Ala Ala

305 310 315 320

Ser Asp Thr Thr Thr Thr Val Ile Leu Ile Trp Val Ile Ser Leu Leu Leu

325 330 335

Asn Asn Ala Asp Val Leu Lys Lys Val Gln Glu Glu Leu Asp Glu Gln

340 345 350

Val Gly Arg Glu Arg Arg Val Glu Glu Ser Asp Ile Ser Asn Leu Pro

355 360 365

Tyr Leu Gln Ala Val Val Lys Glu Thr Met Arg Leu Tyr Pro Pro Ala

370 375 380

Pro Phe Ala Gly Val Arg Ala Phe Ser Glu Asp Cys Thr Val Gly Gly

385 390 395 400

Tyr His Ile Gln Lys Gly Thr Phe Leu Ile Val Asn Leu Trp Lys Leu

405 410 415

His Arg Asp Pro Arg Val Trp Ser Asp Asp Ala Leu Glu Phe Lys Pro

420 425 430

Gln Arg Phe Phe Asp Lys Lys Val Glu Val Lys Gly Gln Asp Phe Glu

435 440 445

Leu Met Pro Phe Gly Gly Gly Arg Arg Met Cys Pro Gly Ser Asn Leu

450 455 460

Gly Met His Met Val His Phe Val Leu Ala Asn Ile Leu Gln Ala Phe

465 470 475 480

Asp Ile Thr Thr Gly Ser Thr Val Asp Met Thr Glu Ser Val Gly Leu

485 490 495

Thr Asn Met Lys Ala Thr Pro Leu Asp Ala Ile Leu Thr Pro Arg Leu

500 505 510

Ser Pro Thr Leu Tyr

515

<210> 2

<211> 495

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (1)..(495)

<223> F6H mutant

<400> 2

Met Ala Met Pro Lys Lys Ser Ser Leu Asn Ala Pro Pro Glu Ala Gly

1 5 10 15

Gly Ala Arg Phe Ile Thr Gly His Leu His Leu Met Asp Gly Arg Ser

20 25 30

Ala Ser Asp Lys Leu Pro His Ile Asn Leu Gly Leu Leu Ala Asp Gln

35 40 45

His Gly Pro Ile Phe Thr Ile Arg Leu Gly Val His Arg Ala Val Val

50 55 60

Val Ser Ser Trp Glu Leu Ala Lys Glu Ile Phe Thr Thr His Asp Thr

65 70 75 80

Ala Val Met Ala Arg Pro Arg Leu Ile Ala Asp Asp Tyr Leu Ser Tyr

85 90 95

Asp Gly Ala Ser Leu Gly Phe Ser Pro Tyr Gly Pro Tyr Trp Arg Glu

100 105 110

Ile Arg Lys Leu Val Thr Thr Glu Leu Leu Ser Ala Arg Arg Ile Glu

115 120 125

Leu Gln Arg Ala Thr Arg Val Arg Glu Ile Thr Gln Phe Thr Gly Glu

130 135 140

Leu Tyr Lys Leu Trp Glu Glu Lys Lys Asp Gly Ser Gly Arg Val Leu

145 150 155 160

Val Asp Met Lys Gln Trp Leu Gly Asn Leu Ser Leu Asn Leu Val Ser

165 170 175

Arg Met Val Val Gly Lys Arg Phe Tyr Gly Gly Asp Asp Ser Glu Thr

180 185 190

Thr Lys Arg Trp Arg Gly Val Met Arg Glu Phe Phe Gln Leu Ile Gly

195 200 205

Gln Phe Ile Pro Gly Asp Gly Leu Pro Phe Leu Arg Trp Leu Asp Leu

210 215 220

Gly Gly Phe Glu Lys Arg Thr Arg Asp Thr Ala Tyr Glu Leu Asp Lys

225 230 235 240

Ile Ile Ala Met Trp Leu Ala Glu Tyr Arg Lys Arg Glu Tyr Ser Gly

245 250 255

Asp Asp Lys Glu Gln Cys Phe Met Ala Leu Met Leu Ser Leu Val Gln

260 265 270

Ala Asn Pro Thr Leu Gln Leu His Tyr Asp Ala Asp Thr Ile Ile Lys

275 280 285

Ala Thr Cys Gln Val Leu Ile Ser Ala Ala Ser Asp Thr Thr Thr Val

290 295 300

Ile Leu Ile Trp Val Ile Ser Leu Leu Leu Asn Asn Ala Asp Val Leu

305 310 315 320

Lys Lys Val Gln Glu Glu Leu Asp Glu Gln Val Gly Arg Glu Arg Arg

325 330 335

Val Glu Glu Ser Asp Ile Ser Asn Leu Pro Tyr Leu Gln Ala Val Val

340 345 350

Lys Glu Thr Met Arg Leu Tyr Pro Pro Ala Pro Phe Ala Gly Val Arg

355 360 365

Ala Phe Ser Glu Asp Cys Thr Val Gly Gly Tyr His Ile Gln Lys Gly

370 375 380

Thr Phe Leu Ile Val Asn Leu Trp Lys Leu His Arg Asp Pro Arg Val

385 390 395 400

Trp Ser Asp Asp Ala Leu Glu Phe Lys Pro Gln Arg Phe Phe Asp Lys

405 410 415

Lys Val Glu Val Lys Gly Gln Asp Phe Glu Leu Met Pro Phe Gly Gly

420 425 430

Gly Arg Arg Met Cys Pro Gly Ser Asn Leu Gly Met His Met Val His

435 440 445

Phe Val Leu Ala Asn Ile Leu Gln Ala Phe Asp Ile Thr Thr Gly Ser

450 455 460

Thr Val Asp Met Thr Glu Ser Val Gly Leu Thr Asn Met Lys Ala Thr

465 470 475 480

Pro Leu Asp Ala Ile Leu Thr Pro Arg Leu Ser Pro Thr Leu Tyr

485 490 495

<210> 3

<211> 501

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (1)..(501)

<223> F6H mutant 8RPtrF6H

<400> 3

Met Ala Leu Leu Leu Ala Val Phe Met Pro Lys Lys Ser Ser Leu Asn

1 5 10 15

Ala Pro Pro Glu Ala Gly Gly Ala Arg Phe Ile Thr Gly His Leu His

20 25 30

Leu Met Asp Gly Arg Ser Ala Ser Asp Lys Leu Pro His Ile Asn Leu

35 40 45

Gly Leu Leu Ala Asp Gln His Gly Pro Ile Phe Thr Ile Arg Leu Gly

50 55 60

Val His Arg Ala Val Val Val Ser Ser Trp Glu Leu Ala Lys Glu Ile

65 70 75 80

Phe Thr Thr His Asp Thr Ala Val Met Ala Arg Pro Arg Leu Ile Ala

85 90 95

Asp Asp Tyr Leu Ser Tyr Asp Gly Ala Ser Leu Gly Phe Ser Pro Tyr

100 105 110

Gly Pro Tyr Trp Arg Glu Ile Arg Lys Leu Val Thr Thr Glu Leu Leu

115 120 125

Ser Ala Arg Arg Ile Glu Leu Gln Arg Ala Thr Arg Val Arg Glu Ile

130 135 140

Thr Gln Phe Thr Gly Glu Leu Tyr Lys Leu Trp Glu Glu Lys Lys Asp

145 150 155 160

Gly Ser Gly Arg Val Leu Val Asp Met Lys Gln Trp Leu Gly Asn Leu

165 170 175

Ser Leu Asn Leu Val Ser Arg Met Val Val Gly Lys Arg Phe Tyr Gly

180 185 190

Gly Asp Asp Ser Glu Thr Thr Lys Arg Trp Arg Gly Val Met Arg Glu

195 200 205

Phe Phe Gln Leu Ile Gly Gln Phe Ile Pro Gly Asp Gly Leu Pro Phe

210 215 220

Leu Arg Trp Leu Asp Leu Gly Gly Phe Glu Lys Arg Thr Arg Asp Thr

225 230 235 240

Ala Tyr Glu Leu Asp Lys Ile Ile Ala Met Trp Leu Ala Glu Tyr Arg

245 250 255

Lys Arg Glu Tyr Ser Gly Asp Asp Lys Glu Gln Cys Phe Met Ala Leu

260 265 270

Met Leu Ser Leu Val Gln Ala Asn Pro Thr Leu Gln Leu His Tyr Asp

275 280 285

Ala Asp Thr Ile Ile Lys Ala Thr Cys Gln Val Leu Ile Ser Ala Ala

290 295 300

Ser Asp Thr Thr Thr Thr Val Ile Leu Ile Trp Val Ile Ser Leu Leu Leu

305 310 315 320

Asn Asn Ala Asp Val Leu Lys Lys Val Gln Glu Glu Leu Asp Glu Gln

325 330 335

Val Gly Arg Glu Arg Arg Val Glu Glu Ser Asp Ile Ser Asn Leu Pro

340 345 350

Tyr Leu Gln Ala Val Val Lys Glu Thr Met Arg Leu Tyr Pro Pro Ala

355 360 365

Pro Phe Ala Gly Val Arg Ala Phe Ser Glu Asp Cys Thr Val Gly Gly

370 375 380

Tyr His Ile Gln Lys Gly Thr Phe Leu Ile Val Asn Leu Trp Lys Leu

385 390 395 400

His Arg Asp Pro Arg Val Trp Ser Asp Asp Ala Leu Glu Phe Lys Pro

405 410 415

Gln Arg Phe Phe Asp Lys Lys Val Glu Val Lys Gly Gln Asp Phe Glu

420 425 430

Leu Met Pro Phe Gly Gly Gly Arg Arg Met Cys Pro Gly Ser Asn Leu

435 440 445

Gly Met His Met Val His Phe Val Leu Ala Asn Ile Leu Gln Ala Phe

450 455 460

Asp Ile Thr Thr Gly Ser Thr Val Asp Met Thr Glu Ser Val Gly Leu

465 470 475 480

Thr Asn Met Lys Ala Thr Pro Leu Asp Ala Ile Leu Thr Pro Arg Leu

485 490 495

Ser Pro Thr Leu Tyr

500

<210> 4

<211> 591

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (1)..(591)

<223> F6H mutant SumotrF6H

<400> 4

Met Ala Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro

1 5 10 15

Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser

20 25 30

Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu

35 40 45

Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg

50 55 60

Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp

65 70 75 80

Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile

85 90 95

Gly Gly Met Pro Lys Lys Ser Ser Leu Asn Ala Pro Pro Glu Ala Gly

100 105 110

Gly Ala Arg Phe Ile Thr Gly His Leu His Leu Met Asp Gly Arg Ser

115 120 125

Ala Ser Asp Lys Leu Pro His Ile Asn Leu Gly Leu Leu Ala Asp Gln

130 135 140

His Gly Pro Ile Phe Thr Ile Arg Leu Gly Val His Arg Ala Val Val

145 150 155 160

Val Ser Ser Trp Glu Leu Ala Lys Glu Ile Phe Thr Thr His Asp Thr

165 170 175

Ala Val Met Ala Arg Pro Arg Leu Ile Ala Asp Asp Tyr Leu Ser Tyr

180 185 190

Asp Gly Ala Ser Leu Gly Phe Ser Pro Tyr Gly Pro Tyr Trp Arg Glu

195 200 205

Ile Arg Lys Leu Val Thr Thr Glu Leu Leu Ser Ala Arg Arg Ile Glu

210 215 220

Leu Gln Arg Ala Thr Arg Val Arg Glu Ile Thr Gln Phe Thr Gly Glu

225 230 235 240

Leu Tyr Lys Leu Trp Glu Glu Lys Lys Asp Gly Ser Gly Arg Val Leu

245 250 255

Val Asp Met Lys Gln Trp Leu Gly Asn Leu Ser Leu Asn Leu Val Ser

260 265 270

Arg Met Val Val Gly Lys Arg Phe Tyr Gly Gly Asp Asp Ser Glu Thr

275 280 285

Thr Lys Arg Trp Arg Gly Val Met Arg Glu Phe Phe Gln Leu Ile Gly

290 295 300

Gln Phe Ile Pro Gly Asp Gly Leu Pro Phe Leu Arg Trp Leu Asp Leu

305 310 315 320

Gly Gly Phe Glu Lys Arg Thr Arg Asp Thr Ala Tyr Glu Leu Asp Lys

325 330 335

Ile Ile Ala Met Trp Leu Ala Glu Tyr Arg Lys Arg Glu Tyr Ser Gly

340 345 350

Asp Asp Lys Glu Gln Cys Phe Met Ala Leu Met Leu Ser Leu Val Gln

355 360 365

Ala Asn Pro Thr Leu Gln Leu His Tyr Asp Ala Asp Thr Ile Ile Lys

370 375 380

Ala Thr Cys Gln Val Leu Ile Ser Ala Ala Ser Asp Thr Thr Thr Val

385 390 395 400

Ile Leu Ile Trp Val Ile Ser Leu Leu Leu Asn Asn Ala Asp Val Leu

405 410 415

Lys Lys Val Gln Glu Glu Leu Asp Glu Gln Val Gly Arg Glu Arg Arg

420 425 430

Val Glu Glu Ser Asp Ile Ser Asn Leu Pro Tyr Leu Gln Ala Val Val

435 440 445

Lys Glu Thr Met Arg Leu Tyr Pro Pro Ala Pro Phe Ala Gly Val Arg

450 455 460

Ala Phe Ser Glu Asp Cys Thr Val Gly Gly Tyr His Ile Gln Lys Gly

465 470 475 480

Thr Phe Leu Ile Val Asn Leu Trp Lys Leu His Arg Asp Pro Arg Val

485 490 495

Trp Ser Asp Asp Ala Leu Glu Phe Lys Pro Gln Arg Phe Phe Asp Lys

500 505 510

Lys Val Glu Val Lys Gly Gln Asp Phe Glu Leu Met Pro Phe Gly Gly

515 520 525

Gly Arg Arg Met Cys Pro Gly Ser Asn Leu Gly Met His Met Val His

530 535 540

Phe Val Leu Ala Asn Ile Leu Gln Ala Phe Asp Ile Thr Thr Gly Ser

545 550 555 560

Thr Val Asp Met Thr Glu Ser Val Gly Leu Thr Asn Met Lys Ala Thr

565 570 575

Pro Leu Asp Ala Ile Leu Thr Pro Arg Leu Ser Pro Thr Leu Tyr

580 585 590

<210> 5

<211> 861

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (1)..(861)

<223> F6H mutant MBPtrF6H

<400> 5

Met Ala Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp

1 5 10 15

Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp

20 25 30

Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys

35 40 45

Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp

50 55 60

Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu

65 70 75 80

Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp

85 90 95

Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val

100 105 110

Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro

115 120 125

Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys

130 135 140

Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp

145 150 155 160

Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly

165 170 175

Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala

180 185 190

Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala

195 200 205

Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr

210 215 220

Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser

225 230 235 240

Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro

245 250 255

Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser

260 265 270

Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr

275 280 285

Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val

290 295 300

Ala Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala

305 310 315 320

Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro

325 330 335

Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala

340 345 350

Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr

355 360 365

Met Pro Lys Lys Ser Ser Leu Asn Ala Pro Pro Glu Ala Gly Gly Ala

370 375 380

Arg Phe Ile Thr Gly His Leu His Leu Met Asp Gly Arg Ser Ala Ser

385 390 395 400

Asp Lys Leu Pro His Ile Asn Leu Gly Leu Leu Ala Asp Gln His Gly

405 410 415

Pro Ile Phe Thr Ile Arg Leu Gly Val His Arg Ala Val Val Val Ser

420 425 430

Ser Trp Glu Leu Ala Lys Glu Ile Phe Thr Thr His Asp Thr Ala Val

435 440 445

Met Ala Arg Pro Arg Leu Ile Ala Asp Asp Tyr Leu Ser Tyr Asp Gly

450 455 460

Ala Ser Leu Gly Phe Ser Pro Tyr Gly Pro Tyr Trp Arg Glu Ile Arg

465 470 475 480

Lys Leu Val Thr Thr Glu Leu Leu Ser Ala Arg Arg Ile Glu Leu Gln

485 490 495

Arg Ala Thr Arg Val Arg Glu Ile Thr Gln Phe Thr Gly Glu Leu Tyr

500 505 510

Lys Leu Trp Glu Glu Lys Lys Asp Gly Ser Gly Arg Val Leu Val Asp

515 520 525

Met Lys Gln Trp Leu Gly Asn Leu Ser Leu Asn Leu Val Ser Arg Met

530 535 540

Val Val Gly Lys Arg Phe Tyr Gly Gly Asp Asp Ser Glu Thr Thr Lys

545 550 555 560

Arg Trp Arg Gly Val Met Arg Glu Phe Phe Gln Leu Ile Gly Gln Phe

565 570 575

Ile Pro Gly Asp Gly Leu Pro Phe Leu Arg Trp Leu Asp Leu Gly Gly

580 585 590

Phe Glu Lys Arg Thr Arg Asp Thr Ala Tyr Glu Leu Asp Lys Ile Ile

595 600 605

Ala Met Trp Leu Ala Glu Tyr Arg Lys Arg Glu Tyr Ser Gly Asp Asp

610 615 620

Lys Glu Gln Cys Phe Met Ala Leu Met Leu Ser Leu Val Gln Ala Asn

625 630 635 640

Pro Thr Leu Gln Leu His Tyr Asp Ala Asp Thr Ile Ile Lys Ala Thr

645 650 655

Cys Gln Val Leu Ile Ser Ala Ala Ser Asp Thr Thr Thr Val Ile Leu

660 665 670

Ile Trp Val Ile Ser Leu Leu Leu Asn Asn Ala Asp Val Leu Lys Lys

675 680 685

Val Gln Glu Glu Leu Asp Glu Gln Val Gly Arg Glu Arg Arg Val Glu

690 695 700

Glu Ser Asp Ile Ser Asn Leu Pro Tyr Leu Gln Ala Val Val Lys Glu

705 710 715 720

Thr Met Arg Leu Tyr Pro Pro Ala Pro Phe Ala Gly Val Arg Ala Phe

725 730 735

Ser Glu Asp Cys Thr Val Gly Gly Tyr His Ile Gln Lys Gly Thr Phe

740 745 750

Leu Ile Val Asn Leu Trp Lys Leu His Arg Asp Pro Arg Val Trp Ser

755 760 765

Asp Asp Ala Leu Glu Phe Lys Pro Gln Arg Phe Phe Asp Lys Lys Val

770 775 780

Glu Val Lys Gly Gln Asp Phe Glu Leu Met Pro Phe Gly Gly Gly Arg

785 790 795 800

Arg Met Cys Pro Gly Ser Asn Leu Gly Met His Met Val His Phe Val

805 810 815

Leu Ala Asn Ile Leu Gln Ala Phe Asp Ile Thr Thr Gly Ser Thr Val

820 825 830

Asp Met Thr Glu Ser Val Gly Leu Thr Asn Met Lys Ala Thr Pro Leu

835 840 845

Asp Ala Ile Leu Thr Pro Arg Leu Ser Pro Thr Leu Tyr

850 855 860

<210> 6

<211> 509

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (1)..(509)

<223> F6H mutant 2B1trF6H

<400> 6

Met Ala Lys Lys Thr Ser Ser Lys Gly Lys Leu Pro Pro Gly Pro Ser

1 5 10 15

Met Pro Lys Lys Ser Ser Leu Asn Ala Pro Pro Glu Ala Gly Gly Ala

20 25 30

Arg Phe Ile Thr Gly His Leu His Leu Met Asp Gly Arg Ser Ala Ser

35 40 45

Asp Lys Leu Pro His Ile Asn Leu Gly Leu Leu Ala Asp Gln His Gly

50 55 60

Pro Ile Phe Thr Ile Arg Leu Gly Val His Arg Ala Val Val Val Ser

65 70 75 80

Ser Trp Glu Leu Ala Lys Glu Ile Phe Thr Thr His Asp Thr Ala Val

85 90 95

Met Ala Arg Pro Arg Leu Ile Ala Asp Asp Tyr Leu Ser Tyr Asp Gly

100 105 110

Ala Ser Leu Gly Phe Ser Pro Tyr Gly Pro Tyr Trp Arg Glu Ile Arg

115 120 125

Lys Leu Val Thr Thr Glu Leu Leu Ser Ala Arg Arg Ile Glu Leu Gln

130 135 140

Arg Ala Thr Arg Val Arg Glu Ile Thr Gln Phe Thr Gly Glu Leu Tyr

145 150 155 160

Lys Leu Trp Glu Glu Lys Lys Asp Gly Ser Gly Arg Val Leu Val Asp

165 170 175

Met Lys Gln Trp Leu Gly Asn Leu Ser Leu Asn Leu Val Ser Arg Met

180 185 190

Val Val Gly Lys Arg Phe Tyr Gly Gly Asp Asp Ser Glu Thr Thr Lys

195 200 205

Arg Trp Arg Gly Val Met Arg Glu Phe Phe Gln Leu Ile Gly Gln Phe

210 215 220

Ile Pro Gly Asp Gly Leu Pro Phe Leu Arg Trp Leu Asp Leu Gly Gly

225 230 235 240

Phe Glu Lys Arg Thr Arg Asp Thr Ala Tyr Glu Leu Asp Lys Ile Ile

245 250 255

Ala Met Trp Leu Ala Glu Tyr Arg Lys Arg Glu Tyr Ser Gly Asp Asp

260 265 270

Lys Glu Gln Cys Phe Met Ala Leu Met Leu Ser Leu Val Gln Ala Asn

275 280 285

Pro Thr Leu Gln Leu His Tyr Asp Ala Asp Thr Ile Ile Lys Ala Thr

290 295 300

Cys Gln Val Leu Ile Ser Ala Ala Ser Asp Thr Thr Thr Val Ile Leu

305 310 315 320

Ile Trp Val Ile Ser Leu Leu Leu Asn Asn Ala Asp Val Leu Lys Lys

325 330 335

Val Gln Glu Glu Leu Asp Glu Gln Val Gly Arg Glu Arg Arg Val Glu

340 345 350

Glu Ser Asp Ile Ser Asn Leu Pro Tyr Leu Gln Ala Val Val Lys Glu

355 360 365

Thr Met Arg Leu Tyr Pro Pro Ala Pro Phe Ala Gly Val Arg Ala Phe

370 375 380

Ser Glu Asp Cys Thr Val Gly Gly Tyr His Ile Gln Lys Gly Thr Phe

385 390 395 400

Leu Ile Val Asn Leu Trp Lys Leu His Arg Asp Pro Arg Val Trp Ser

405 410 415

Asp Asp Ala Leu Glu Phe Lys Pro Gln Arg Phe Phe Asp Lys Lys Val

420 425 430

Glu Val Lys Gly Gln Asp Phe Glu Leu Met Pro Phe Gly Gly Gly Arg

435 440 445

Arg Met Cys Pro Gly Ser Asn Leu Gly Met His Met Val His Phe Val

450 455 460

Leu Ala Asn Ile Leu Gln Ala Phe Asp Ile Thr Thr Gly Ser Thr Val

465 470 475 480

Asp Met Thr Glu Ser Val Gly Leu Thr Asn Met Lys Ala Thr Pro Leu

485 490 495

Asp Ala Ile Leu Thr Pro Arg Leu Ser Pro Thr Leu Tyr

500 505

<210> 7

<211> 712

<212> PRT

<213> Arabidopsis thaliana

<400> 7

Met Ser Ser Ser Ser Ser Ser Ser Ser Thr Ser Met Ile Asp Leu Met Ala

1 5 10 15

Ala Ile Ile Lys Gly Glu Pro Val Ile Val Ser Asp Pro Ala Asn Ala

20 25 30

Ser Ala Tyr Glu Ser Val Ala Ala Glu Leu Ser Ser Met Leu Ile Glu

35 40 45

Asn Arg Gln Phe Ala Met Ile Val Thr Thr Ser Ile Ala Val Leu Ile

50 55 60

Gly Cys Ile Val Met Leu Val Trp Arg Arg Ser Gly Ser Gly Asn Ser

65 70 75 80

Lys Arg Val Glu Pro Leu Lys Pro Leu Val Ile Lys Pro Arg Glu Glu

85 90 95

Glu Ile Asp Asp Gly Arg Lys Lys Val Thr Ile Phe Phe Gly Thr Gln

100 105 110

Thr Gly Thr Ala Glu Gly Phe Ala Lys Ala Leu Gly Glu Glu Ala Lys

115 120 125

Ala Arg Tyr Glu Lys Thr Arg Phe Lys Ile Val Asp Leu Asp Asp Tyr

130 135 140

Ala Ala Asp Asp Asp Glu Tyr Glu Glu Lys Leu Lys Lys Glu Asp Val

145 150 155 160

Ala Phe Phe Phe Leu Ala Thr Tyr Gly Asp Gly Glu Pro Thr Asp Asn

165 170 175

Ala Ala Arg Phe Tyr Lys Trp Phe Thr Glu Gly Asn Asp Arg Gly Glu

180 185 190

Trp Leu Lys Asn Leu Lys Tyr Gly Val Phe Gly Leu Gly Asn Arg Gln

195 200 205

Tyr Glu His Phe Asn Lys Val Ala Lys Val Val Asp Asp Ile Leu Val

210 215 220

Glu Gln Gly Ala Gln Arg Leu Val Gln Val Gly Leu Gly Asp Asp Asp

225 230 235 240

Gln Cys Ile Glu Asp Asp Phe Thr Ala Trp Arg Glu Ala Leu Trp Pro

245 250 255

Glu Leu Asp Thr Ile Leu Arg Glu Glu Gly Asp Thr Ala Val Ala Thr

260 265 270

Pro Tyr Thr Ala Ala Val Leu Glu Tyr Arg Val Ser Ile His Asp Ser

275 280 285

Glu Asp Ala Lys Phe Asn Asp Ile Asn Met Ala Asn Gly Asn Gly Tyr

290 295 300

Thr Val Phe Asp Ala Gln His Pro Tyr Lys Ala Asn Val Ala Val Lys

305 310 315 320

Arg Glu Leu His Thr Pro Glu Ser Asp Arg Ser Cys Ile His Leu Glu

325 330 335

Phe Asp Ile Ala Gly Ser Gly Leu Thr Tyr Glu Thr Gly Asp His Val

340 345 350

Gly Val Leu Cys Asp Asn Leu Ser Glu Thr Val Asp Glu Ala Leu Arg

355 360 365

Leu Leu Asp Met Ser Pro Asp Thr Tyr Phe Ser Leu His Ala Glu Lys

370 375 380

Glu Asp Gly Thr Pro Ile Ser Ser Ser Ser Leu Pro Pro Pro Phe Pro Pro

385 390 395 400

Cys Asn Leu Arg Thr Ala Leu Thr Arg Tyr Ala Cys Leu Leu Ser Ser

405 410 415

Pro Lys Lys Ser Ala Leu Val Ala Leu Ala Ala His Ala Ser Asp Pro

420 425 430

Thr Glu Ala Glu Arg Leu Lys His Leu Ala Ser Pro Ala Gly Lys Val

435 440 445

Asp Glu Tyr Ser Lys Trp Val Val Glu Ser Gln Arg Ser Leu Leu Glu

450 455 460

Val Met Ala Glu Phe Pro Ser Ala Lys Pro Pro Leu Gly Val Phe Phe

465 470 475 480

Ala Gly Val Ala Pro Arg Leu Gln Pro Arg Phe Tyr Ser Ile Ser Ser

485 490 495

Ser Pro Lys Ile Ala Glu Thr Arg Ile His Val Thr Cys Ala Leu Val

500 505 510

Tyr Glu Lys Met Pro Thr Gly Arg Ile His Lys Gly Val Cys Ser Thr

515 520 525

Trp Met Lys Asn Ala Val Pro Tyr Glu Lys Ser Glu Asn Cys Ser Ser

530 535 540

Ala Pro Ile Phe Val Arg Gln Ser Asn Phe Lys Leu Pro Ser Asp Ser

545 550 555 560

Lys Val Pro Ile Ile Met Ile Gly Pro Gly Thr Gly Leu Ala Pro Phe

565 570 575

Arg Gly Phe Leu Gln Glu Arg Leu Ala Leu Val Glu Ser Gly Val Glu

580 585 590

Leu Gly Pro Ser Val Leu Phe Phe Gly Cys Arg Asn Arg Arg Met Asp

595 600 605

Phe Ile Tyr Glu Glu Glu Leu Gln Arg Phe Val Glu Ser Gly Ala Leu

610 615 620

Ala Glu Leu Ser Val Ala Phe Ser Arg Glu Gly Pro Thr Lys Glu Tyr

625 630 635 640

Val Gln His Lys Met Met Asp Lys Ala Ser Asp Ile Trp Asn Met Ile

645 650 655

Ser Gln Gly Ala Tyr Leu Tyr Val Cys Gly Asp Ala Lys Gly Met Ala

660 665 670

Arg Asp Val His Arg Ser Leu His Thr Ile Ala Gln Glu Gln Gly Ser

675 680 685

Met Asp Ser Thr Lys Ala Glu Gly Phe Val Lys Asn Leu Gln Thr Ser

690 695 700

Gly Arg Tyr Leu Arg Asp Val Trp

705 710

<210> 8

<211> 641

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (1)..(641)

<223> AtCPR mutant trAtCPR

<400> 8

Met Arg Arg Ser Gly Ser Gly Asn Ser Lys Arg Val Glu Pro Leu Lys

1 5 10 15

Pro Leu Val Ile Lys Pro Arg Glu Glu Glu Ile Asp Asp Gly Arg Lys

20 25 30

Lys Val Thr Ile Phe Phe Gly Thr Gln Thr Gly Thr Ala Glu Gly Phe

35 40 45

Ala Lys Ala Leu Gly Glu Glu Ala Lys Ala Arg Tyr Glu Lys Thr Arg

50 55 60

Phe Lys Ile Val Asp Leu Asp Asp Tyr Ala Ala Asp Asp Asp Glu Tyr

65 70 75 80

Glu Glu Lys Leu Lys Lys Glu Asp Val Ala Phe Phe Phe Leu Ala Thr

85 90 95

Tyr Gly Asp Gly Glu Pro Thr Asp Asn Ala Ala Arg Phe Tyr Lys Trp

100 105 110

Phe Thr Glu Gly Asn Asp Arg Gly Glu Trp Leu Lys Asn Leu Lys Tyr

115 120 125

Gly Val Phe Gly Leu Gly Asn Arg Gln Tyr Glu His Phe Asn Lys Val

130 135 140

Ala Lys Val Val Asp Asp Ile Leu Val Glu Gln Gly Ala Gln Arg Leu

145 150 155 160

Val Gln Val Gly Leu Gly Asp Asp Asp Gln Cys Ile Glu Asp Asp Phe

165 170 175

Thr Ala Trp Arg Glu Ala Leu Trp Pro Glu Leu Asp Thr Ile Leu Arg

180 185 190

Glu Glu Gly Asp Thr Ala Val Ala Thr Pro Tyr Thr Ala Ala Val Leu

195 200 205

Glu Tyr Arg Val Ser Ile His Asp Ser Glu Asp Ala Lys Phe Asn Asp

210 215 220

Ile Asn Met Ala Asn Gly Asn Gly Tyr Thr Val Phe Asp Ala Gln His

225 230 235 240

Pro Tyr Lys Ala Asn Val Ala Val Lys Arg Glu Leu His Thr Pro Glu

245 250 255

Ser Asp Arg Ser Cys Ile His Leu Glu Phe Asp Ile Ala Gly Ser Gly

260 265 270

Leu Thr Tyr Glu Thr Gly Asp His Val Gly Val Leu Cys Asp Asn Leu

275 280 285

Ser Glu Thr Val Asp Glu Ala Leu Arg Leu Leu Asp Met Ser Pro Asp

290 295 300

Thr Tyr Phe Ser Leu His Ala Glu Lys Glu Asp Gly Thr Pro Ile Ser

305 310 315 320

Ser Ser Leu Pro Pro Pro Phe Pro Pro Cys Asn Leu Arg Thr Ala Leu

325 330 335

Thr Arg Tyr Ala Cys Leu Leu Ser Ser Pro Lys Lys Ser Ala Leu Val

340 345 350

Ala Leu Ala Ala His Ala Ser Asp Pro Thr Glu Ala Glu Arg Leu Lys

355 360 365

His Leu Ala Ser Pro Ala Gly Lys Val Asp Glu Tyr Ser Lys Trp Val

370 375 380

Val Glu Ser Gln Arg Ser Leu Leu Glu Val Met Ala Glu Phe Pro Ser

385 390 395 400

Ala Lys Pro Pro Leu Gly Val Phe Phe Ala Gly Val Ala Pro Arg Leu

405 410 415

Gln Pro Arg Phe Tyr Ser Ile Ser Ser Ser Ser Pro Lys Ile Ala Glu Thr

420 425 430

Arg Ile His Val Thr Cys Ala Leu Val Tyr Glu Lys Met Pro Thr Gly

435 440 445

Arg Ile His Lys Gly Val Cys Ser Thr Trp Met Lys Asn Ala Val Pro

450 455 460

Tyr Glu Lys Ser Glu Asn Cys Ser Ser Ala Pro Ile Phe Val Arg Gln

465 470 475 480

Ser Asn Phe Lys Leu Pro Ser Asp Ser Lys Val Pro Ile Ile Met Ile

485 490 495

Gly Pro Gly Thr Gly Leu Ala Pro Phe Arg Gly Phe Leu Gln Glu Arg

500 505 510

Leu Ala Leu Val Glu Ser Gly Val Glu Leu Gly Pro Ser Val Leu Phe

515 520 525

Phe Gly Cys Arg Asn Arg Arg Met Asp Phe Ile Tyr Glu Glu Glu Leu

530 535 540

Gln Arg Phe Val Glu Ser Gly Ala Leu Ala Glu Leu Ser Val Ala Phe

545 550 555 560

Ser Arg Glu Gly Pro Thr Lys Glu Tyr Val Gln His Lys Met Met Asp

565 570 575

Lys Ala Ser Asp Ile Trp Asn Met Ile Ser Gln Gly Ala Tyr Leu Tyr

580 585 590

Val Cys Gly Asp Ala Lys Gly Met Ala Arg Asp Val His Arg Ser Leu

595 600 605

His Thr Ile Ala Gln Glu Gln Gly Ser Met Asp Ser Thr Lys Ala Glu

610 615 620

Gly Phe Val Lys Asn Leu Gln Thr Ser Gly Arg Tyr Leu Arg Asp Val

625 630 635 640

Trp

<210> 9

<211> 25

<212>DNA

<213> Primer

<400> 9

tataccatgg aactgagcag tgtga 25

<210> 10

<211> 36

<212>DNA

<213> Primer

<400> 10

ctcgaattcg gatccactag tttaatataa agtcgg 36

<210> 11

<211> 43

<212>DNA

<213> Primer

<400> 11

ctttaagaag gagatatacc atggcgatgc cgaagaaaag ctc 43

<210> 12

<211> 63

<212>DNA

<213> Primer

<400> 12

ctttaagaag gagatatacc atggctctgt tattagcagt ttttatgccg aagaaaagct 60

ctt 63

<210> 13

<211> 39

<212>DNA

<213> Primer

<400> 13

ctttaagaag gagatatacc atggctaaaa tcgaagaag 39

<210> 14

<211> 35

<212>DNA

<213> Primer

<400> 14

ctgaaagacg cgcagactat gccgaagaaa agctc 35

<210> 15

<211> 35

<212>DNA

<213> Primer

<400> 15

gagcttttct tcggcatagt ctgcgcgtct ttcag 35

<210> 16

<211> 87

<212>DNA

<213> Primer

<400> 16

ctttaagaag gagatatacc atggctaaga aaacgagctc taaagggaag ctcccaccag 60

gacctagcat gccgaagaaa agctctt 87

<210> 17

<211> 44

<212>DNA

<213> Primer

<400> 17

ctttaagaag gagatatacc atggcggact cagaagtcaa tctt 44

<210> 18

<211> 36

<212>DNA

<213> Primer

<400> 18

gagaacagat tggtggtatg ccgaagaaaa gctctt 36

<210> 19

<211> 36

<212>DNA

<213> Primer

<400> 19

aagagctttt cttcggcata ccaccaatct gttctc 36

<210> 20

<211> 27

<212>DNA

<213> Primer

<400> 20

tataccatgg gtgactgcgt tgccccg 27

<210> 21

<211> 30

<212>DNA

<213> Primer

<400> 21

cgggatcctt acttcggcag gtcgccgctc 30

<210> 22

<211> 29

<212>DNA

<213> Primer

<400> 22

cgggatccct tatgcgactc ctgcattag 29

<210> 23

<211> 26

<212>DNA

<213> Primer

<400> 23

gcccaagctt ttatgccagc atcttc 26

<210> 24

<211> 31

<212>DNA

<213> Primer

<400> 24

agatatacat atggttacgg tggaagaata c 31

<210> 25

<211> 30

<212>DNA

<213> Primer

<400> 25

ccgctcgagt taggtagcca cactatgcag 30

<210> 26

<211> 30

<212>DNA

<213> Primer

<400> 26

ccgctcgagc tagaaataat tttgtttaac 30

<210> 27

<211> 28

<212> DNA

<213> Primer

<400> 27

gagcctaggt tagttaccga ttttaaag 28

<210> 28

<211> 38

<212>DNA

<213> Primer

<400> 28

gaagatgctg gcataaaagc ttcgatcccg cgaaatta 38

<210> 29

<211> 38

<212>DNA

<213> Primer

<400> 29

cgacttaagc attatgcggc cgcctacgcc aggttttc 38

Claims (10)

1. A method for producing baicalein, comprising:

introducing into a host cell a gene expressing a flavone 6-hydroxylase and a cytochrome P450 oxidoreductase; the flavone 6-hydroxylase is modified mutant flavone 6-hydroxylase, and the amino acid sequence of the flavone 6-hydroxylase is shown as SEQ ID NO. 6; the cytochrome P450 oxidoreductase is a wild type enzyme or an engineered mutant enzyme, the amino acid sequence of the wild type enzyme is shown as SEQ ID NO. 7, and the amino acid sequence of the mutant enzyme is shown as SEQ ID NO. 8; the host cell is an E.coli cell;

culturing the recombinant host cell in a culture system containing chrysin, thereby producing baicalein.

2. A method for converting chrysin into baicalein is characterized in that flavone 6-hydroxylase and cytochrome P450 oxidoreductase are used for catalyzing chrysin, so that a hydroxyl group is added into the structure of chrysin to generate baicalein; the flavone 6-hydroxylase is modified mutant flavone 6-hydroxylase, and the amino acid sequence of the flavone 6-hydroxylase is shown as SEQ ID NO. 6; the cytochrome P450 oxidoreductase is a wild type enzyme or an engineered mutant enzyme, the amino acid sequence of the wild type enzyme is shown as SEQ ID NO. 7, and the amino acid sequence of the mutant enzyme is shown as SEQ ID NO. 8.

3. Use of a recombinant host cell comprising exogenous genes expressing a flavone 6-hydroxylase and a cytochrome P450 oxidoreductase in the production of baicalein; the flavone 6-hydroxylase is modified mutant flavone 6-hydroxylase, and the amino acid sequence of the flavone 6-hydroxylase is shown as SEQ ID NO. 6; the cytochrome P450 oxidoreductase is a wild type enzyme or an engineered mutant enzyme, the amino acid sequence of the wild type enzyme is shown as SEQ ID NO. 7, and the amino acid sequence of the mutant enzyme is shown as SEQ ID NO. 8; the host cell is an E.coli cell.

4. A method of preparing a host cell for the production of baicalein comprising: introducing a gene expressing flavone 6-hydroxylase and cytochrome P450 oxidoreductase into a host cell to obtain a recombinant strain; the flavone 6-hydroxylase is modified mutant flavone 6-hydroxylase, and the amino acid sequence of the flavone 6-hydroxylase is shown as SEQ ID NO. 6; the cytochrome P450 oxidoreductase is a wild type enzyme or an engineered mutant enzyme, the amino acid sequence of the wild type enzyme is shown as SEQ ID NO. 7, and the amino acid sequence of the mutant enzyme is shown as SEQ ID NO. 8; the host cell is an E.coli cell.

5. Use of a kit comprising recombinant host cells in the production of baicalein; the recombinant host cell comprises exogenous genes for expressing flavone 6-hydroxylase and cytochrome P450 oxidoreductase; the flavone 6-hydroxylase is modified mutant flavone 6-hydroxylase, and the amino acid sequence of the flavone 6-hydroxylase is shown as SEQ ID NO. 6; the cytochrome P450 oxidoreductase is a wild type enzyme or an engineered mutant enzyme, the amino acid sequence of the wild type enzyme is shown as SEQ ID NO. 7, and the amino acid sequence of the mutant enzyme is shown as SEQ ID NO. 8; the host cell is an E.coli cell.

6. The application of mutant flavone 6-hydroxylase and cytochrome P450 oxidoreductase in the production of baicalein is that the mutant flavone 6-hydroxylase is modified mutant flavone 6-hydroxylase, and the amino acid sequence of the mutant flavone 6-hydroxylase is shown as SEQ ID NO. 6; the cytochrome P450 oxidoreductase is a wild type or mutant enzyme, the amino acid sequence of the wild type enzyme is shown as SEQ ID NO. 7, and the amino acid of the mutant enzyme is shown as SEQ ID NO. 8.

7. The use according to claim 6, wherein the mutant flavone 6-hydroxylase and cytochrome P450 oxidoreductase form a fusion polypeptide.

8. The use according to claim 6, wherein the polynucleotide encoding the mutant flavone 6-hydroxylase and cytochrome P450 oxidoreductase is introduced into a host cell to produce baicalein; the host cell is an E.coli cell.

9. The use according to claim 8, wherein an expression construct is prepared comprising a polynucleotide encoding the mutant flavone 6-hydroxylase and cytochrome P450 oxidoreductase, introduced into a host cell.

10. The use of claim 6, wherein said producing baicalein is by adding a hydroxyl group to the structure of chrysin to form baicalein.